IMPROVED SPECTROPHOTOMETRIC ASSAY FOR BETA-LACTAM RESIDUES IN KIDNEY TISSUE

This paper describes a detection system for beta-lactams using a comme rcially prepared carboxypeptidase enzyme (CPase) and a substrate syste m in which lactic acid is cleaved from a synthetic peptide, N alpha-N epsilon-diacetyl-L-lysyl-d-alanyl-d-lactic acid. The lactate is itself oxidized by lactate dehydrogenase to form NADH. Oxidized NAD(+) is re generated by diaphorase with the simultaneous reduction of the colourl ess 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride h ydrate (INT) indicator substrate to produce a red-mauve colour that is proportional to CPase activity. The presence of beta-lactams decrease s the intensity of colour produced. The lower limit of detection for b enzyl penicillin (Pen G) by this system is 20 ng g(-1) compared with 5 0 ng g(-1) by the same assay but using an R-d-ala-d-ala substrate from a commercial kit.